The tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA), and epidermal growth factor (EGF) inhibit growth of human A431 epidermoid carcinoma cells within 24 to 48 hours after exposure of the cells to these agents. Addition of TPA and EGF inhibit cell growth in an additive or synergistic manner. These effects on cell growth are preceded by a change in the activity of a calcium dependent, cyclic nucleotide-independent and phospholipid-dependent protein kinase (protein kinase C). Specifically, EGF produced a 2- to 3-fold stimulation in protein kinase C activity within 30 to 60 minutes following exposure to the cells. TPA alone had no effect on protein kinase C activity. However, TPA attenuated the increase in protein kinase C activity that was induced by EGF. In EGF treated cells (250 ng/ml, one hour) there was a three to four-fold increase in the phosphorylation of a cytosolic protein at 17-20 Kd (pp17-20, pI approximately 5.5) and a moderate increase in the phosphorylation of other proteins at molecular weights of 27,40,45 and 70-80 Kd as detected by two-dimensional gel electrophoresis. Treatment of the cells with TPA 10 to the-7, one hour) resulted in a similar effect on the phosphorylation of pp17-20 as well as on pp27 and pp70-80. However, TPA in contrast to EGF did not affect the phosphorylation of pp40 and pp45. In combination with EGF, TPA attenuated the EGF-induced phosphorylation of pp40, but did not affect the phosphorylation of the other proteins. In vitro studies demonstrated three bands following one-dimensional gel electrophoresis at 17, 26, and 47 Kd which are phosphorylated in the presence of phospholipids and might therefore be substrates for protein kinase C and modulated by EGF.